Various methods of adding fermentable substrates to fermentation media for bioconversion are known to those skilled in the art. The most common of these are adding the fermentable substrate in solution, as a dry powder or a powder in aqueous suspension. These methods usually provide substrate concentrations of about 0.5-20 g./l.
The addition of fermentable substrates to the fermentation medium in an organic water-miscible solvent is well known to those skilled in the art. For example, methanol or acetone may be used, see Tetrahedron 18, 581 (1962) at page 487. Ethyl alcohol has been used to add Reichstein's Compound S for fermentation, see J.A.C.S. 75, 5369 (1953) and Applied Microbiology 3, 16 (1955). Various lower aliphatic acid amides and the N-alkyl derivatives thereof, in particular DMF (N,N-dimethylformamide), have been used to add various steroids to fermentation media, see U.S. Pat. No. 3,138,541 and Applied Microbiology 7, 276 (1959).
In U.S. Pat. No. 3,770,586 the 21-hydroxy steroids are added in the form of a 21-hydrocarbon dicarboxylic acid ester alkali metal salt. This salt has increased water solubility and therefore higher effective substrate concentrations (2-15 g./l.) are obtained.
Alternatively, the fermentable substrate can be added to the aqueous fermentation medium in a powdered form. Progesterone has been added in this manner, see I and EC Process Design and Development 5, 285 (1966). When the progesterone was added in a finely divided particle size obtained by grinding, efficient bioconversions at substrate concentrations of 20-50 g./l. were obtained, see Applied Microbiology 8, 345 (1960). An alternative method of adding a powdered fermentable substrate to fermentation medium is by micronization. Here the substrate (progesterone) is comminuted which allows for efficient bioconversion at substrate concentrations in the range of 16-20 g./l., see U.S. Pat. No. 3,201,324.
U.S. Pat. No. 3,840,570 discloses a process for the preparation of sterols from plant sources, especially tall oil pitch, by extraction with a water-alcohol-hydrocarbon mixture followed by saponification and subsequent purification. A. H. Conner, et al. in Applied and Environmental Microbiology 32, 310 (1976) describe the microbial conversion of tall oil sterols and crude sitosterol from soy beans to androstenedione-type compounds using DMF as the solvent for introducing sterols to the fermentation. The process of the present invention generates a concentrated aqueous suspension of very finely divided or microcrystalline sterols by dissolving sterols from a sterol source in a volatile organic diluent, addition of the solution to an aqueous system, and rapid removal of the organic diluent.
While the most common method utilized for adding fermentable substrates to fermentation media involves dissolving the fermentable substrate in an aqueous miscible organic diluent and adding that diluent to the fermentation medium, this process has the problem that due to the toxicity of the organic diluents to the microorganisms in the fermentation medium, the concentration of the aqueous miscible organic diluent must be kept low. Since the concentration of the aqueous miscible organic diluent must be kept low, the amount of substrate which can be added to the fermentation will of course be limited (usually less than 2 g./l.).
The problem with adding powders to the fermentation medium is that they tend to clump and not disperse well. Sterols tend to be waxy and do not undergo milling very well, hence it is difficult to obtain finely ground powders.
W. C. McGregor et al. Biotechnol, Bioeng. 14, 831 (1972) described a pilot plant bioconversion of a steroid using a resting cell suspension. Several methods of introducing the substrate for bioconversion were attempted including adding the steroid dissolved in methylene chloride. The present invention differs from the McGregor process in that (1) the sterol or sterols in an organic diluent are added to an aqueous system prior to inoculation with the microorganism and (2) the organic diluent is removed producing very finely divided or microcrystalline sterols. Additionally, McGregor et al. indicate that adding the steroid in methylene chloride was not very satisfactory, see page 835, while the method of the present invention produces excellent results.
S. I. Ladinskaya et al. in Gidroliz. Lesokhim Prom. 24, 7 (1971) and O. I. Andreeva et al. ibid, 24, 28 (1971) disclose methods of obtaining crystalline phytosterols from a sterol-ethanol solution by addition of water. An alcohol-water suspension of crystals is totally unsuitable for fermentation. The present invention produces an aqueous suspension of very finely divided or microcrystalline sterols which is suitable for fermentation.
The process of the present invention is a method for adding a sterol or sterols to an aqueous system for bioconversion which differs from prior processes in that the material to be fermented is added to the aqueous system in an organic diluent which is removed from the aqueous system by heat and/or reduced pressure. Upon removal of the organic diluent an aqueous suspension of very finely divided or microcrystalline sterols results which is suitable for fermentation. The present invention permits the addition of large amounts of fermentable substrate to the fermentation medium without the retention of a toxic organic diluent. Additionally, the process of the present invention provides substrate concentration as high as 200 g./l.
Processes for the preparation of a source of sterols for bioconversion have become all the more important now that a process has been developed to cleave the side chain of sterols to produce androsta-1,4-diene-3,17-dione and androst-4-ene-3,17-dione, see U.S. Pat. Nos. 3,684,657 and 3,759,791. Note especially, that the examples in U.S. Pat. No. 3,759,791 show that sterols from various sources may be utilized for conversion to androst-4-ene-3,17-dione and androsta-1,4-diene-3,17-dione.